Arginine, which is one of essential amino acids for the cell growth of many species, could be synthesized from citrulline in the normal cells through a two-step reaction catalyzed by argininosuccinate synthetase (ASS) and argininosuccinate lyase (AL) shown as following: L-arginine+H2OL-citrulline+NH3. Therefore, normal cells don't require external supply of arginine for growth. However, the nature of arginine auxotrophy appears in some abnormal appearing cells, such as hepatocellular carcinomas (HCC), melanoma or other sarcomas, within which arginine could not be synthesized because of lack of ASS in these cells. Therefore, these kinds of abnormal appearing cells could be survived only in the environment containing arginine which is the main essential amino acid required in the growth of these abnormal cells. In the presence of enzymes for degradation of arginine, the arginine existing in the environment could be eliminated which will result in starvation of these abnormal cells whose growth will be significantly inhibited. Therefore, the arginine-degrading enzyme can be used as a potential clinical drug for liver cancer, melanoma and other diseases. Arginine deiminase (ADI), which catalyzes the conversion of arginine to citrulline, could be used for eliminating arginine. The pADI isolated from Pseudomonas putida could effectively kill the tumor cells in vitro (Jones J B, The effect of arginine deiminase on murine leukemia lymphoblasts (Ph.D. dissertation), Oklahoma City, Okla., University of Oklahoma, 1981), particularly tumor cells relevant to liver cancer and melanoma. However, the pADI isolated from Pseudomonas putida failed to demonstrate its role in vivo. According to research results, the possible reasons of this about this phenomenon include that pADI almost had no bioactivity at neutral pH, and meanwhile the pADI isolated from Pseudomonas putida had strong immunogenicity to experimental animals and was easy to induce autoantibody synthesis after entering into the organism, and the resulted antigen-antibody immune complexes could be quickly eliminated from the blood circulation of experimental animals.
Takaku, et al (Haruo Takaku et al., In vivo anti-tumor activity of arginine deiminase purified from Mycoplasma arginini, Int J Cancer., 51:244-249, 1992) isolated another arginine deiminase (aADI) from Mycoplasma arginini. Unlike the pADI which was isolated from Pseudomonas putida, aADI showed the highest activity at pH of 6.0-7.5 and was very stable at neutral pH. However, pADI and ADI, which were both derived from lower microorganism species, still show solid antigenicity and thus were easy to be cleared by the circulatory system in human body.
After chemical modification (such as PEG, gelatin, polysaccharides), they can effectively block the epitope on the surface of protein, and reduce or eliminate the inherent immunogenicity of the protein, and the molecular weight of the modified protein was increased, which can extend the internal clearance rate and increase its half-life, so protein modification was a preferred method for solving the immunogenicity. Polyethylene glycol (PEG) has been recognized as a safe protein chemical modification reagent and some PEG-modified drugs have been used in clinical practice. However, PEG-modified proteins, including arginine deiminase, L-asparaginase and other enzymes will lead to a decline in or even complete loss of enzyme activity (Mehvar R, Modulation of the pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation, J. Pharm Pharmaceut Sci., 3:125-136, 2000). When Holtsberg F W et al (Holtsberg F W et al., Poly(ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties, J Control Release. 80:259-271,2002) studied the PEG-modified ADI, they found that when the ADI was combined with 8-10 PEG20000 molecules, only less than 50% of its enzyme activity was retained, and after further modification, when more than 20 PEG molecules were combined, its enzyme activity was almost entirely lost. Therefore, for this kind of proteins, it is necessary to optimize and balance between the activity retention and protein PEG modification in order to achieve the clinical requirements of the drugs.